Enantioseparation of N-fluorenylmethoxycarbonyl α-amino acids on polysaccharide-derived chiral stationary phases by reverse mode liquid chromatography

Abstract

The enantioseparation of N-protected fluorenylmethoxycarbonyl ( N-FMOC) α-amino acids was carried out on three polysaccharide-derived chiral stationary phases, such as cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD), amylose tris(3,5-dimethyl-phenylcarbamate) (Chiralpak AD) and cellulose tris(4-methylbenzoate) (Chiralcel OJ), and the influence of acetonitrile composition and pH of the eluents on the enantioseparation in reverse mode chromatography was examined. The best separation of the enantiomers was achieved with 40% acetonitrile in 50 mM phosphate buffer at pH 2. However, increasing the composition of acetonitrile to 50% on Chiralcel OD yielded a considerable decrease of retention time with minimum loss of resolution. The elution order of N-FMOC α-amino acid enantiomers on Chiralcel OD and OJ were quite different, indicating that both phases could be used in a complementary manner for the separation of the enantiomers of N-FMOC α-amino acids. The positive relationship between the capacity factor of N-FMOC α-amino acids and the hydrophobicity of amino acids indicated that hydrophobicity plays an important role on the retention of the N-FMOC α-amino acids in the reverse mode.